(sar&#39; 1&#39;,thr&#39; 8&#39;+9 angiotensin ii as an angiotensin antagonist

ABSTRACT

A new octapeptide having profound angiotensin antagonist properties has been found. The new compound resembles angiotensin II except for the two terminal amino acids, carrying an unnatural amino acid at the N-terminus.

United States Patent [191 Bumpus et al.

[ [SARKTHRUANGIOTENSIN II AS AN ANGIOTENSIN ANTAGONIST 22 Filed: May 10,1974 211 Appl No.: 468,853

[52] US. Cl 260/1115; 424/177 [51] Int. Cl. C07C 103/52; A61K 37/00 [58]Field of Search 260/1 12.5

1 Dec. 9, 1975 [56] References Cited UNITED STATES PATENTS 3,816,3856/1974 Gillessen et a] 260/1125 Primary Examiner-Lewis Gotts AssistantExaminer-Reginald J Suyat Attorney, Agent, or FirmPaul D. Burgauer;Robert L. Niblack [57] ABSTRACT A new octapeptide having profoundangiotensin antagonist properties has been found. The new compoundresembles angiotensin 11 except for the two ter minal amino acids,carrying an unnatural amino acid at the N-terminus.

1 Claim, N0 Drawings 1 [SARKTHRQANGIOTENSIN ll AS AN ANGIOTENSINANTAGONIST DETAILED DESCRlPTlON OF THE INVENTIONHNCHCOHNCHCONCHCO-HNCHCOOH The peptide of this invention possessesvaluable 3S pharmacological activity. It is capable of inhibiting thepressor effect of angiotensin II. Thus, when administered by intravenousinfusion into rats in a very small amount, the pressor effect ofangiotensin ll is inhibited. By virtue of this inhibitory property uponangiotensin ll induced blood pressure elevation, the peptide of thisinvention is a valuable agent for counteracting hypertension due toangiotensin ll. [t is also capable of reducing blood pressure in acuteunilateral renal hypertensive rats upon intravenous infusion.

The octapeptide of this invention is readily prepared in accordance withknown methods for preparing peptides. Such methods involve the buildingof a linear chain of amino acids through repetitive amide linkagesemploying in such sequential alignment the necessary protective groupssusceptible to ready removal by conventional cleavage methods which donot affect the peptide bonds. The adaptation of such methods to thepeptide of this invention is described in the example below. All theamino acids used in the above structure of Formula I are in theL-configuration. The corresponding chain with D-amino acids does notshow the pharmacological effect described below.

In order to illustrate the method for making the compound of structure1, reference is made to the following example, which however, is notintended to limit the invention in any respect. In this example, theabbreviation BOC- is used in its accepted meaning, referring to tertiarybutyloxycarbonyl. It also should be understood that all amino acids usedin this example are in their L- configuration. The BOC- amino acids usedbelow are either commercially available or were prepared according tothe method of Schwyzer et al., Helv. Chim. Acta 42, 2622 (1959).

EXAMPLE A solution of 3.09 g of BOC-(O-henzyll-threonine and 1.4 ml oftriethylamine in 50 ml of ethylacetate was added to 10 g ofchloromethylpolystyrene/divinylbenzene (98:2) copolymer of a mesh sizebetween 200 and 400, containing 5.02% chlorine. The mixture was stirredat 80C for 36 hours. The esterified polymer was filtered, washed severaltimes in sequence with ethanol, dilute acetic acid, water, ethanol andmethanol. The polymer was dried in vacuo over phosphorous pentoxide.Hydrolysis of an aliquot of the polymer and subsequent amino acidanalysis indicated that O.ll millimoles of BOC-(O-benzyl)-threonine wereesterified per gram of the polymer. Further coupling ofthe BOC- proline,BOC-N-imidazolebenzyl-histidine, BOC-isoleucine,BOC-(O-benzyl)-tyrosine, BOC-valine. BOC- nitroarginine andBOC-sarcosine in the respective order was carried out by utilizing theaction given below for each amino acid residue. Unless specified, allwashings were carried out three times for 3 minutes each, first withglacial acetic acid and second with methylene chloride. The BOC groupwas removed by treatment with 40% (volume/volume) of trifluoroaceticacid in methylene chloride for 30 minutes, preceded by a prewash withthis reagent for 3 minutes to avoid dilution of the trifluoroacetic acidby the previous methylene chloride wash. The deprotected amino acidpolymer ester was washed five times for 3 minutes each with chloroformand the trifluoroacetate salt neutralized by treating the residue for 7minutes with 10% triethylamine in chloroform, followed by three 3 minutewashings with chloroform and methylene chloride in sequence. Thesubsequent incoming BOC-amino acid was added in a 2-fold excess inmethylene chloride and the mixture was stirred for 10 minutes. In thecase of BOC-nitroarginine and BOC-benzyl histidine, these 0 materialswere first dissolved in dimethylformamide,

' fold excess and mixing allowed for 8 hours. The polymer-peptide chainwas then washed with DMF followed 0 by a washing with DMF/methylenechloride (1:1) and the coupling step with the BOC-amino acid and DCI wasrepeated using a mixture of lzl DMF/methylene chloride as the solvent.The polymer chain was then washed with methanol to removedicyclohexylurea and finally washed with DMF. Completeness of eachcoupling at intermediate stages was checked by known color reactiontests. The apparatus used for the above synthesis was of the manual typedescribed by Khosla, Smeby and Bumpus, Science 156,253 (1967). Allcouplings were carried out at 0 5C to avoid racemization;l-hydroxybenzotriazole was used as an additive to minimize raeemizationof histidine during the coupling of BOC-imidazolebenzyl histidine [seeG. C. Windridge and E. C. Jorgensen, J.A.C.S., 93, 6318 (1971)].

In the above sequence, the blocked (protected) amino acids were coupledin sequence to the O-benzyl threonine-polymer, using BOC-proline, BOC-N-3 imidazole-benzyl-histidine, BOC-isoleucine, BOC-(O- benzyl)-tyrosine,BOC-valine and BOC-nitroarginine and BOC-sarcosine to produce a peptideof the structure of formula I with amino acids 1 8 bound to the polymersubstrate.

The protected peptide polymer was suspended in approximately 100 ml offreshly distilled trifluoroacetic acid and a slow stream of hydrogenbromide, prewashed with 10% resorcinol in acetic acid, was passedthrough the suspension under anhydrous conditions for about 30 minuteswith occasional shaking. The suspension was filtered and the polymer waswashed with trifluoroacetic acid. The combined filtrates were evaporatedat room temperature in vacuo. The amorphous powder was washed withether, dissolved in a mixture of methanol/acetic acid/water 5:l:1 andthe solution hydrogenated at 3.5 kg/cm over 0.5 parts of palladium blackper part of peptide weight for 48 hours with shaking. The product waspurified on a 2.5 X l cm column of Sephadex G 25 (a partiallycross-linked dextran gel having an exclusion of molecular weight sizesof 5000, marketed by Pharmacia of Uppsala, Sweden) usingn-butanol/acetic acid/water/pyridine (l:3:l2:l0) as the developingsolvent. Fractions in column chromatography were cut without regard foryield to obtain the desired compound in the pure form and no attempt wasmade to rechromatograph the minor fractions for identification purposes.The average yield of the product obtained in this manner varied between40 and 60% based on the millimoles of C-terminal amino acid esterifiedonto the polymer. The homogeneity of the compound was determined by thethin layer chromatography in various solvent systems of different pH,electrophoresis at pH 1.95 and 8.6 and amino acid analysis. proving thatthe compound is homogeneous with R; 0.09 (n-butanol/acetic acid/water 4:I25 upperphase) and R, 0.50 (n-butanol/ethylacetate/acetic acid/waterl:l:l:l), R, 0.04 (nbutanolIpyridine/water l0'.l:5), R, 0.46(n-butanol/acetic acid/water/pyridine l 5:3: [2: on cellulose thinlayerplates. The chemical analysis showed that the required amino acids werepresent in the expected ratio.

The above antagonist was studied both in vitro and in vivo. The in vitroassay mainly evolves around an assay of isolated rabbit aortic stripsmounted in a muscle bath in 5 ml of Krebs solution.

The strips were placed under 2 grams of passive tension and allowed toequilibrate for L5 2 hours. Cumulative dose-response curves wereobtained for an giotensin [I at concentrations of l to 64 ng/ml. Theantagonist was added to the bath at a certain concentration, allowed toequilibrate for 5 minutes and the response to angiotensin II againtested. The various doses of the antagonist thus tested were 1, 10, 100and i000 ng/ml. Changes in these curves were plotted and measurement ofshift to the right of the dose response curve were measured. At the sametime, it was assured that the maximal response of the tissue remainedunchanged. At the end of the experimental period the antagonist waswashed out and cumulative dose-response curves were repeated again.Results of these studies are expressed in the form of pA values asdefined by O. Arunlakshna and H. O. Schild, Brit, J. Pharmacol. l4, 48(1959 The compound for formula I showed a pA value of 8.79 i 0.14.

[n the in vivo assays, rats were anesthetized with sodium amytal andfurther treated with 0.6 mg atropine and pentolinium tartrate. They werevagotomized and direct recording of the blood pressure was measuredthrough the carotid artery, while the femoral artery was used forinfusing the desired dose over a period of 30 minutes in a physiologicalsaline solution.

The dose-response curves of angiotensin ll at 0.9 to 54 ng/kg/min. werecompared by linear regression analysis with those of the antagonist atdoses of l, 10, and 1000 ng/kg/min. Infusion of the above antagonistinto rats thus prepared led to the initial transient pressor response of0.60 on a scale set artificially at 100 for angiotensin ll using thesame system.

For determining its antagonistic properties the compound was infused inrats at a dose level of 250, 500, 1250 ng/kg/min. The blocking effect ofthe compound was calculated from the dose-response curve of angiotensinll before and during the infusion of the analog. General procedureadopted for this assay has been described by Bumpus et al., Circ. Res.32-33 (Supplement l), [-l50 (1973). The compound blocked the pressorresponse of angiotensin [I at a dose level of 250 ng/kg/min or greater.

Since the above compound is particularly suitable for injection orinfusion, it is particularly valuable that the compound iswater-soluble. A suitable dosage unit can be prepared by simplydissolving the above compound in water or physiological saline at aconcentration of between 50 and 6000 ng/ml. Such a solution can beadministered directly or it can be stored under proper condition forperiods of several weeks without deterioration. particularly whencombined with l 5% of a preservative such as benzyl alcohol and/or isbufiered to a suitable pH with a nontoxic, pharmaceutically acceptablebuffer. A commonly employed buffer for an injectable solution isTris(hydroxymethyl)aminomethane but simple salts such as sodiumphosphate or acetate can be used. Preferably, the vehicle or medium inwhich the compound of formula I is dissolved for an injectable orinfusable solution is buffered to a pH of 7 to 7.5.

The duration of action of the compound can be prolonged by injecting anoil solution intramuscularly. Oils suitable for this purpose are codliver oil, sesame oil, refined coconut oil, etc. Antagonistic effectsare thus observed for 24 hours or longer after a single i.m. injectionof the drug dissolved in such an oil.

What is claimed is:

l. The octapeptide Sar-L-Arg-L-Val-L-Tyr-L-lle-L- His-L-Pro-L-Thr.

1. THE OCTAPEPTIDE SAR-L-ARG-L-VAL-L-TYR-L-IIE-L-HIS-LPRO-L-THR.